Chromatography methods typically rely on exploitation of a solid surface that bears at least one chemical functionality to actively engage in interactions with biomolecules in order to sort the components of a complex sample according to that chemical functionality. These methods are called adsorptive chromatography methods. Surface chemistries differ among adsorptive methods, along with the chemical means to release bound components, but the operational format is the same. One example is bioaffinity chromatography, in which an inert surface is substituted with a biological ligand specific for a component of interest from a complex sample. The component of interest binds, the rest do not, and the component of interest is subsequently released by changing the chemical conditions. Ion exchange chromatography is representative of a greater diversity of methods. An inert surface is substituted covalently with a charged chemical group. In the case of anion exchange chromatography, the chemical group is positively charged. Sample components of sufficient negative charge bind, with the most negatively charged binding most strongly. Sample components lacking sufficient negative charge fail to bind. Bound components can be released in order of the strength of their interaction by the application of an increasing gradient of salt, which imposes a gradually increasing degree of disruption to the charge interactions, releasing the bound components in order of the intensity of their interaction with the anon exchanger. Except for differences of chemistry, the same operational format is applied to cation exchange chromatography, hydrophobic interaction chromatography, reversed-phase chromatography, and numerous examples of so-called mixed-mode chromatography, including hydroxyapatite.
An exception to the adsorptive methods reliance upon chemical functionality is size exclusion chromatography (SEC), also known as gel filtration and gel permeation chromatography. Chemical interactions between molecules and the surface of the media in such applications are effectively nil. SEC works by the differential diffusion of sample components into the pores of particles packed in a column. Very large components are excluded from the pores and pass only through the interparticle space. (Arbitrarily) mid-sized molecules diffuse into the larger pores. This gives them access to a larger fluid volume that the excluded molecules that have access to only the interparticle space. (Arbitrarily) small molecules may diffuse into all of the pores, which gives them access to a yet greater fluid volume. The greater the fluid volume with which a given size class of molecules is in equilibrium, the larger the volume of fluid required to displace them from the column. Thus large molecules elute first from SEC columns, followed in order by molecules of decreasing size.
Precipitation methods are well-known throughout the field of biology, including for purification of proteins and viruses. Two of the most common methods, so-called salt precipitation and PEG precipitation, exploit a force known as preferential exclusion. The term began to be used widely in the early 1980s and has since become the accepted terminology for describing the interactions of dissolved molecules (solutes) with proteins [1-5]. Preferentially excluded solutes interact with proteins in such a way that it leaves the proteins surrounded by a layer of water that is deficient in the preferentially excluded solute. This solute-deficient zone is referred to as the zone of preferential hydration, or preferential hydration shell, or sheath. When preferentially hydrated proteins encounter one another in solution, their preferential hydration sheaths merge. The higher the concentration of the excluded solute, the more strongly the two proteins remain associated. So-called kosmotropic salts, such as ammonium sulfate, sodium citrate, and potassium phosphate are strongly excluded from protein surfaces. Nonionic organic polymers such as polyethylene glycol (PEG) are also known to be strongly excluded from protein surfaces [3-5]. These precipitation methods are performed by dissolving large amounts of the preferentially excluded agent in a sample containing the species to be precipitated. As the preferentially excluded agent ascends toward a threshold level, it causes target species that randomly contact one another to remain associated by sharing the water from their respective preferential hydration sheaths. This leads to formation of large insoluble aggregations that eventually precipitate, after which they can be recovered by centrifugation or filtration. Precipitation methods however impose undesirable limitations, chief of which is that purification performance is recognized as universally inferior to chromatography methods, and also suffers from a high degree of variability from batch to batch, especially in conjunction with variations in the concentration of the product to be precipitated. Recovery is likewise variable, and can be prohibitively low when the product of interest is present at low concentrations. These issues are known to derive from the dependency of precipitation methods on the interactions of highly heterogeneous surfaces, namely the proteins being precipitated. Commercial applications persist, but most have been replaced by chromatography because the latter usually offers better recovery and a higher degree of purity. Interest in precipitation methods remains however because they potentially offer higher productivity than chromatography, the materials are cheaper, and the equipment is simpler to operate.
The force of preferential exclusion has been used to enhance existing interactions between proteins and the surfaces of adsorptive chromatography media. Salts are well known to enhance binding in hydrophobic interaction chromatography (HIC). This is the standard way in which binding is achieved with this technique. They have also been used to enhance binding with some affinity methods, such as protein A affinity chromatography (6). Ammonium sulfate was reported to cause binding of proteins to a non-functionalized porous particle chromatography support in the 1970s (7) but was not pursued, possibly due to limitations in the method of sample preparation and application to the column. Direct addition of excess ammonium sulfate to a sample creates precipitates that clog chromatography columns. Addition of non-precipitating amounts of salt would be expected to support low capacities, as apparently experienced in reference 7.
The force of preferential exclusion has also been used to enhance binding of adsorptive chromatography methods with nonionic polymers such as PEG. In contrast to salts, it does not work with HIC because the inherent hydrophobicity of the PEG interferes directly with the binding mechanism. PEG can be used to precipitate proteins inside a HIC column, and the proteins can be subsequently resolubilized, but this is not adsorptive chromatography and it lacks utility: capacity and resolution are prohibitively limited [6]. PEG has been used to enhance binding with affinity chromatography [6], ion exchange chromatography [8], and hydroxyapatite chromatography [9], and it is known to interfere with the separation achieved in SEC [10]. In all these cases it retards elution, especially of large molecules such as aggregates, which sometimes aids in their separation [9], but its use is discouraged by its high viscosity. High viscosity is a particular problem for all methods of chromatography on porous particles because they depend on diffusion to transport proteins to, within, and from the pores. Diffusivity is directly proportional to viscosity, so column performance drops in direct proportion to the PEG concentration. Viscosity also increases shear forces that occur in the interparticle space of chromatography columns. These limitations are tolerated in some instances because PEG has been shown to more strongly affect the binding of large molecules in comparison to small one, thus enhancing the ability of hydroxyapatite to improve fractionation of antibody aggregates from nonaggregated antibody [7,8]. Since both liabilities increase in direct proportion to PEG concentration, it is not surprising that its broader application has been avoided.
Fluidized particle beds provide an alternative to chromatography in packed beds. In fluidized beds, particles are dispersed throughout the sample containing the target molecule. After binding the target molecule, the particles are concentrated, so that unbound contaminants can be washed away, and so that the bound product can be eluted at a high concentration. Concentration of the particles can be achieved by various methods. Particles with density greater than water can be sedimented. Iron-core particles can be concentrated in a magnetic field. Particles may also be concentrated on filtration membranes. Particle size may vary from less than 100 nm to more than 100 microns. The surface of particles for expanded bed chromatography is normally functionalized with chemical groups that interact strongly with the intended target molecule. For example, many publications report the immobilization of protein A on fluidized particles to capture and purify IgG. Other publications describe functionalization with charged groups, hydrophobic groups, metal affinity groups, and groups employing multiple chemistries. These various functionalizations allow fluidized bed formats to offer the same range of chemical selectivities offered by same the functionalizations in fixed bed chromatography methods.
The importance of fluidized beds is that they offer the physical handling characteristics to overcome one of the most serious limitations of packed beds: low productivity. Traditional porous particle columns require slow flow rates that impose excessive overall process time intervals. These time intervals are multiplied when the chromatography media are so expensive as to force users to run multiple cycles on a reduced volume column because the price of sufficient chromatography material to run a process in a single cycle is prohibitive. However, for any method to fulfill the needs of initial product capture from crude biological samples, also requires that it be able to achieve good binding capacity under near physiological pH and salt concentration. Every known chromatography method has been evaluated for this application, but only bioaffinity chromatography has fulfilled this requirement to date. All others require substantial modification of conditions and/or are rendered ineffective by components of cell culture supernatants.